A loss of parvalbumin-containing interneurons is associated with diminished oscillatory activity in...

A loss of parvalbumin-containing interneurons is associated withdiminished oscillatory activity in an animal model ofschizophrenia

Daniel J. Lodge1, Margarita M. Behrens2, and Anthony A. Grace11Departments of Neuroscience, Psychiatry, and Psychology, University of Pittsburgh, Pittsburgh,PA, 15260, USA2Department of Medicine, Division of Geriatrics, University of California San Diego, La Jolla, CA92093-0746

AbstractDecreased GABAergic signaling is among the more robust pathologies observed post-mortem inschizophrenia; however, the functional consequences of this deficit are still largely unknown. Herewe demonstrate, in a verified animal model of schizophrenia, that a reduced expression ofparvalbumin- (PV) containing interneurons is correlated with a reduction in coordinated neuronalactivity during task performance in freely moving rats. More specifically, methylazoxymethanolacetate (MAM)-treated rats display a decreased density of parvalbumin positive interneuronsthroughout the medial prefrontal cortex (mPFC) and ventral (but not dorsal) subiculum of thehippocampus. Furthermore, the reduction in interneuron functionality is correlated with asignificantly reduced gamma- band response to a conditioned tone during a latent inhibitionparadigm. Finally, deficits in mPFC and ventral hippocampal oscillatory activity are associated withan impaired behavioral expression of latent inhibition in MAM-treated rats. Thus, we propose thata decrease in intrinsic GABAergic signaling may be responsible, at least in part, for the prefrontaland hippocampal hypofunctionality observed during task performance, which is consistentlyobserved in animal models as well as in schizophrenia in humans. In addition, a deficit in intrinsicGABAergic signaling may be the origin of the hippocampal hyperactivity purported to underlie thedopamine dysfunction in psychosis. Such information is central to gaining a better understanding ofthe disease pathophysiology and alternate pharmacotherapeutic approaches.

Keywordsparvalbumin; GABA; hippocampus; prefrontal cortex; schizophrenia; MAM

IntroductionAmong the most robust pathologies observed in schizophrenia is a decrease in GABAergicsignaling (Perry et al., 1979; Benes and Berretta, 2001; Heckers et al., 2002; Reynolds et al.,2002; Lewis et al., 2005; Benes et al., 2007; Lisman et al., 2008). Specifically, a decrease inglutamic acid decarboxylase (GAD)-1 mRNA and GAD-67 protein is observed post-mortemthroughout the cortex of schizophrenia patients (Akbarian et al., 1995; Volk et al., 2000;Hashimoto et al., 2003). These GABA deficits are largely restricted to the class of GABAergic

Correspondence: D.J. Lodge, Department of Neuroscience, University of Pittsburgh, A210 Langley Hall, Pittsburgh, PA 15260, USA.Ph: (412) 624-7332, Fax: (412) 624-9198, [email protected].

NIH Public AccessAuthor ManuscriptJ Neurosci. Author manuscript; available in PMC 2009 September 30.

Published in final edited form as:J Neurosci. 2009 February 25; 29(8): 2344–2354. doi:10.1523/JNEUROSCI.5419-08.2009.

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interneurons containing the calcium binding protein parvalbumin (Lewis et al., 2005). Theseneurons synapse on the cell body or axon initial segment of glutamatergic neurons and thusare positioned to potently regulate pyramidal cell output. Furthermore, it is likely that areduction in PV interneuron functionality would result not only in a decreased inhibitorycontrol over pyramidal cell activity, but also a reduction in the coordinated activity of largebrain networks. Indeed, substantial data demonstrate the importance of fast-spikinginterneurons in the generation of cortical and hippocampal oscillatory potentials (Buzsaki etal., 1983; Whittington et al., 1995; Tamas et al., 2000; Bartos et al., 2002; Bartos et al.,2007; Freund and Katona, 2007).

A failure in coordinated information processing between brain regions has been suggested toaccount for a wide range of deficits in schizophrenia, including both positive and cognitivesymptoms (Haig et al., 2000; Ford et al., 2002; Gonzalez-Hernandez et al., 2003; Spencer etal., 2003; Schmiedt et al., 2005; Cho et al., 2006; Uhlhaas et al., 2006; Basar-Eroglu et al.,2007; Uhlhaas et al., 2008). Furthermore, theta and gamma oscillations, induced in theprefrontal cortex during cognitive tasks, are reduced in schizophrenia patients, whoconsequently perform poorly on these tasks (Gonzalez-Hernandez et al., 2003; Schmiedt et al.,2005; Cho et al., 2006; Basar-Eroglu et al., 2007). Given evidence of a GABA dysfunction inschizophrenia patients and an association between GABA interneurons and coordinatedneuronal activity, we examined the role of PV-containing interneurons in the regulation ofsynchronized mPFC and vHipp activity during task performance in an animal model ofschizophrenia, namely the MAM G17 model.

The MAM G17 model that we first developed (Grace and Moore, 1998) employs theadministration of a mitotoxin, methylazoxymethanol acetate (MAM), on gestational day (GD)17 to pregnant rats to induce a developmental disruption (Grace and Moore, 1998; Talaminiet al., 2000; Gourevitch et al., 2004; Le Pen et al., 2006; Moore et al., 2006). This modelrecapitulates a pathodevelopmental process leading to schizophrenia-like phenotypes in rodentoffspring, which include anatomical changes (Moore et al., 2006; Penschuck et al., 2006),behavioral deficits (Grace and Moore, 1998; Talamini et al., 2000; Flagstad et al., 2004;Gourevitch et al., 2004; Moore et al., 2006) and altered neuronal information processing (Lavinet al., 2005; Goto and Grace, 2006; Lodge and Grace, 2007). Using this model, we nowdemonstrate that a decrease in intrinsic GABAergic functionality is associated with adiminished gamma band response during task performance in freely moving rats.

Materials & MethodsAll experiments were performed in accordance with the guidelines outlined in the USPHSGuide for the Care and Use of Laboratory Animals and were approved by the InstitutionalAnimal Care and Use Committee of the University of Pittsburgh.

AnimalsMAM-treated rats were prepared as described previously (Moore et al., 2006; Lodge and Grace,2007). In brief, timed pregnant female Sprague Dawley rats were obtained at gestational day(GD) 15 and housed individually in plastic breeding tubs. MAM (diluted in saline, 20mg/kg,i.p.) was administered on GD17. Control dams received injections of saline (1ml/kg i.p). Malepups were weaned on day 21 and housed in groups of 2–3 with litter mates until ~ 12 weeksof age, at which time they were used for physiological or anatomical studies. All experimentswere performed on multiple litters of MAM- and saline-treated rats.

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ImmunohistochemistryImmunohistochemical studies were performed as described previously using commerciallyavailable antibodies (Behrens et al., 2007). Adult male rats were anaesthetized with sodiumpentobarbital (60 mg/kg i.p.) and perfused transcardially with saline (50 ml) followed byparaformaldehyde (100 ml: 4 % w/v in 0.1 M PBS). Rats were decapitated and their brainsremoved, post-fixed for 24 hours (4 % w/v PFA in 0.1 M PBS), and stored in 0.1 M PBS untilsectioning. Coronal sections (50 µm) were obtained using a Vibratome. Sequential sectionscomprising the prefrontal region (Bregma +5.0 to +2.0), dorsal (Bregma −1.5 to −4.5) andventral hippocampus (Bregma −4.5 to −7.5) (6 slices per region) were used in fluorescencedouble-immunohistochemistry to detect expression of GAD67 and parvalbumin as described(Behrens et al., 2007). Briefly, slices were treated with 1 % sodium-borohydride for 15 minprior to blocking in 10 % normal goat serum (NGS) for 16 h at 4 °C. Primary antibodies (anti-GAD67 monoclonal 1:2000 (Millipore: MAB5406)) and anti-parvalbumin 1:3000 (Swant:PV28)) were applied in 2 % NGS for 24 h followed by AlexaFluor conjugated goat anti-rabbit(568) or goat anti-mouse (488) antibodies for 1 hr at room temperature and mountedsequentially on glass slides using Vectashield.

Confocal microscopy and image analysisMounted slices were evaluated for fluorescence under settings for the AlexaFuor dyes 488 (Ex488, Em 500–530 band path) and 568 (Ex 543, Em 560 long path) on a LSM510 Metamultiphoton laser confocal microscope equipped with Argon and HeNe lasers and a 545 beamsplitter, and using a 10X and 40X water immersion objectives. The setting of the confocalmicroscope were kept constant throughout the imaging such that fluorescence intensities couldbe compared between animals and imaging days. (Laser powers: 488 = 21%; 543 = 18%.PMT(488): Gain 855 V; offset: 0, PMT(543): Gain 775 V; offset: 0) For slice imaging, each slicewas imaged across the prefrontal and anterior and ventral hippocampal regions (two imagesper slice). Six slices were imaged per animal. For fluorescence intensity quantification a z-stack of 8 images was obtained (corresponding to 1.4 µm on the z-axis) using MetaMorphanalysis software. All PV-neurons in the images were analyzed for their parvalbumin andGAD67 content. For counting PV- and non-PV-interneurons, slices were imaged every 5 µmacross the entire slice, and then collapsed to obtain one image of the resulting z-stack (area:921.5 µm2). All PV-positive cells as well as all GAD67 positive cells in the images werecounted. Since all PV-positive also contain GAD, the cell numbers reported for GAD67 areonly those GAD67 positive cells that did not express detectable levels of PV. The density ofneurons/mm3 for each animal region was obtained by calculating the number of neurons/sliceusing Abercrombie’s correction (Abercrombie, 1946) and a neuron diameter of 12 µm. Slicepreparation and imaging was performed by a researcher unaware of treatment groups.

Survival SurgeryAll survival surgical procedures were performed under general anesthesia in a semi-sterileenvironment. Briefly, male rats (275–350g) were anesthetized with sodium pentobarbital(60mg/kg i.p.) and placed in a stereotaxic apparatus using blunt atraumatic ear bars. Customlength polyimide-insulated stainless steel wire electrodes (Plastics One: MS303/2-AIU) werelowered into the right medial prefrontal cortex (A/P +3.2 mm from bregma, M/L −0.5 mmfrom midline, D/V −4.0 mm ventral of skull surface) and right ventral hippocampus (A/P −6.0mm from bregma, M/L −5.3 mm from midline, D/V −7.0 mm ventral of skull surface). Non-insulated stainless steel ground wires were wrapped around a skull screw. The electrodes werefixed in place with dental cement and a total of five anchor screws. Once the cement wascompletely solid, the wound was sutured, the rat removed from the stereotaxic frame andmonitored closely until conscious. Rats received antibiotic treatment (gentamicin 3mg/kg s.c.)and post-operative analgesia (Children’s Tylenol syrup in softened rat chow; 5% v/w) ad

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libitum for 48 hours. Rats were housed with a reverse light/dark cycle (lights on 1900 – 0700)for at least two weeks before conducting behavioral experiments.

Latent InhibitionAll behavioral testing was performed during the active period of the diurnal cycle (between0700 and 1900). A representation of the behavioral paradigm is presented in Fig 1. Rats werebrought to the testing area and the chronically implanted intracranial electrodes were connectedto a CyberAmp 320 preamplifier (Axon Instruments) via a four channel commutator (PlasticsOne) and the rats placed in an open field arena (Coulbourn Instruments) where baselineoscillatory activity was recorded for a period of 5 mins. The output of the preamplifier wasamplified (×1000), filtered (low pass: 1kHz) and digitized (1kHz) for offline analysis. Ratswere divided into 2 groups, with one group pre-exposed to a 2 second tone for 50 trials with apseudorandom inter-trial interval (range 15 – 45 seconds) and tone-evoked changes in localfield potentials were recorded. The other group had no tone presentation but LFPs wererecorded for 50 trials. All rats were subsequently exposed to a standard auditory fearconditioning procedure that involved a 2 second tone presentation that co-terminated for thefinal second with a mild (0.6mA) footshock, administered through stainless steel bars in thefloor of the chamber (Coulbourn Instruments). Conditioned pairings were performed 10 timeswith a pseudorandom intertrial interval (45 – 100 seconds). Rats were then returned to theanimal colony room. 24 hours following conditioning, rats were placed in the conditioningchamber where LFP responses and spontaneous locomotor activity in the X-Y plane wasmonitored for 5 mins by beam breaks and recorded with TruScan software (CoulbournInstruments). All rats were then exposed to 10 presentations of a 2 second tone with apseudorandom intertrial interval (45 – 100 seconds) where locomotion (for a period of 5 mins)and tone-evoked LFP responses were recorded.

HistologyAt the cessation of the experiments all rats were killed by a lethal dose of anesthetic (sodiumpentobarbital, 120mg/kg i.p.). Rats were decapitated and their brains removed, fixed for at least48 hours (8% w/v paraformaldehyde in phosphate buffered saline: PBS), and cryoprotected(25% w/v sucrose in PBS) until saturated. Brains were sectioned (60µm coronal sections),mounted onto gelatin-chrome alum coated slides and stained with Cresyl violet forhistochemical verification of electrode sites. All histology was performed with reference to astereotaxic atlas and electrode locations are presented in Fig 2 (Paxinos and Watson, 1986).

Electrophysiology AnalysisAnalysis of spontaneous local field potential activity was performed using AutoSignal software(Seasolve Software Inc.). In brief, LFP oscillatory activity was filtered (0–58Hz), detrended(mean subtracted and linear trend removed) and analysis across time was performed on a fourminute period of spontaneous recording using a continuous wavelet (Morlet) time-frequencyanalysis that reported integrated power (Time-Integral Squared Amplitude, TISA) across time.In addition, 2 second representative epochs were examined using a Fourier time-domainreconstruction to graphically demonstrate the principle frequency components of the LPFsignal, whereas 10 second epochs were Fourier-filtered to quantify cortical oscillations in thetheta (4–12 Hz) and gamma (30–55Hz) bands. Evoked changes in LPF responses wereexamined using chronux analysis routines (www.chronux.org) in MATLAB (TheMathWorks). In brief, continuous process multi-taper time-frequency spectral analyses wereperformed on auditory-evoked LPF signals. Tone-evoked changes in power were comparedusing peak analyses (GraphPad Prism) and the maximum increases in power following the tonewere determined for each rat and are expressed as a % of baseline. All data are represented asthe mean ± standard error of the mean (SEM) unless otherwise stated.

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http://www.chronux.org/

MaterialsMethyl azoxymethanol acetate (MAM) was purchased from Midwest Research Institute(Kansas City, MO) and Pentobarbital sodium (USP) was obtained from OvationPharmaceuticals Inc (Deerfield, IL). Mouse monoclonal anti-GAD67 was obtained fromMillipore, and rabbit anti-parvalbumin was obtained from Swant. All other chemicals andreagents were of either analytical or laboratory grade and purchased from various suppliers.

ResultsImmunohistochemistry

Rats that received GD 17 MAM injections exhibited a regionally selective reduction in thedensity of PV positive neurons throughout the mPFC (Fig 3a/c - SAL: 1712 ± 62 PV-cells/mm3, MAM: 1226.9 ± 29 PV-cells/mm3, n=5, p<0.001 1-way ANOVA, Tukey post-hoc) andventral subiculum of hippocampus (Fig 3b/c SAL: 2346.3 ± 146 cells/mm3, MAM: 1255.4 ±96 cells/mm3, n=4, p<0.001 1-way ANOVA, Tukey post-hoc) with no significant differencesin the number of GAD-67 positive/parvalbumin negative neurons in either the mPFC (Fig 3a/d SAL: 2267 +/− 211 cells/mm3, MAM: 2117 +/− 152 cells/mm3, n=5) or ventral subiculumof hippocampus (Fig 3b/d SAL: 5019 +/− 633 cells/mm3, MAM: 4249 +/− 273 cells/mm3,n=4). These differences in PV cell density were not observed in the dorsal subiculum of thehippocampus (Fig 3c - SAL: 1870.4 ± 198.6 PV-cells/mm3, MAM: 2087.6 ± 124.7 PV-cells/mm3, n=5 p=0.381) while a small decrease in PV cell density was observed throughout theanterior cingulate cortex (Fig 3c - SAL: 2133.4 ± 91 PV-cells/mm3, MAM: 1775.7 ± 83 PV-cells/mm3 n=5, p=0.02 1-way ANOVA, Tukey post-hoc). In addition, there were no significantdifferences in fluorescence intensity for either GAD-67 (mPFC (arbitrary units)- SAL: 44.0 ±2.3 , MAM: 52.3 ± 3.6 ; vSub- SAL: 43.5 ± 2.5, MAM: 42.1 ± 3.0, n=4–5) or PV (mPFC-SAL: 190.2 ± 8.6, MAM: 185.7 ± 5.2; vSub- SAL: 88.0 ± 9.1, MAM: 72.7 ± 2.0, n=4–5).

Spontaneous LPF ActivitySpontaneous oscillatory activity was observed in baseline recordings from the mPFC andvHipp of both MAM- and saline-treated rats (Fig 4,5). Continuous wavelet time-frequencyspectral analysis demonstrated a predominant peak in the theta range (peak ~7.5Hz) and asecondary peak in the delta range (peak ~2Hz) throughout the vHipp of both saline- (Fig 4a,b)and MAM- (Fig 4c,d) treated rats. In contrast, mPFC local field potentials displayedsignificantly less oscillatory activity (Fig 5) with a prominent low frequency peak in the deltarange (<3Hz), a secondary peak in the theta range (peak ~7.5Hz) and a tertiary high-frequencypeak in the beta/gamma range (peak ~20Hz) in both saline- (Fig 5a,b) and MAM- (Fig 5c,d)treated rats. Interestingly, there were no significant differences between MAM and saline ratsin spontaneous activity throughout the mPFC (theta (PSD-TISA: µV2.Hz−1)- SAL: 92.7 ± 11.7,MAM: 102.9 ± 13.5 ; gamma (PSD-TISA: µV2.Hz−1)- SAL: 27.5 ± 3.9, MAM: 34.9 ± 7.6,n=9–10) or vHipp (theta (PSD-TISA: µV2.Hz−1)- SAL: 649.5 ± 154.0, MAM: 433.4 ± 156.9 ;gamma (PSD-TISA: µV2.Hz−1)- SAL: 186.4 ± 79.8, MAM: 77.92 ± 18.7, n=9–10); however,high between subject variability, due to electrode location, may mask any differences betweensaline and MAM-treated rats.

Latent InhibitionRats were prepared for behavioral testing and simultaneous evoked field recordings (Fig 1).Consistent with previous literature in untreated rats, the presentation of a conditioned toneevoked a significant decrease in locomotor activity in both saline- (Fig 6a; Baseline: 920.3 ±37.0 cm, Tone: 164.3 ± 23.0 cm, n=4, p<0.05 2-way RM ANOVA with Holm-Sidak post hoc)and MAM-(Fig 6b; Baseline: 960.2 ± 33.8 cm, Tone: 403.2 ± 108.7 cm, n=5, p<0.05 2-wayRM ANOVA with Holm-Sidak post hoc) treated rats. Control rats that had been exposed to

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the tone prior to the conditioning period displayed robust latent inhibition; i.e. an attenuatedconditioned response to the tone (Fig 6a; No Tone pre-exposure: 164.3 ± 23.0 cm, Tone pre-exposure: 385.6 ± 81.9 cm, n=4, p<0.05 2-way RM ANOVA with Holm-Sidak post hoc). Incontrast and consistent with previous data, MAM-treated rats displayed a deficit in latentinhibition, with previous exposure to the tone having no significant effect on the subsequentlocomotor response to the conditioned stimulus (Fig 6b; No Tone pre-exposure: 403.2 ± 108.7cm, Tone pre-exposure: 520.8 ± 74.6 cm, n=5, ns 2-way RM ANOVA). It should be noted thatMAM-treated rats display a reduced response to the conditioned tone when compared to salinerats that is not associated with alterations in auditory processing since MAM treated rats displaya lower threshold for responding to auditory stimuli (Moore et al., 2006).

Evoked LPF ActivityElectrophysiological recordings from the mPFC and vHipp were performed in freely-behavingrats during latent inhibition training and testing. A representative trace of tone-evoked activityis presented in Supplementary Figure 1. Multi-tapered spectral analysis of tone-evoked activitydemonstrated a slight response to the tone presentation prior to conditioning. In both salineand MAM-treated rats, the unconditioned tone tended to increase mPFC theta (Fig 7a,b; Sal148.6 ± 16.8, MAM 129.9 ± 15.8 % of baseline), mPFC gamma (Fig 9a,b; Sal 124.6 ± 14.3,MAM 115.5 ± 5.5 % of baseline) and vHipp gamma (Fig 10a,b; Sal 142.7 ± 18.1, MAM 123.2± 11.3 % of baseline) with no observable change in vHipp theta (Fig 8a,b; Sal 106.5 ± 6.5,MAM 110.4 ± 9.7 % of baseline). Interestingly, following fear conditioning in saline-treatedrats, the conditioned tone induced a massive increase in mPFC theta (Fig 7c; 237.8 ± 36.3 %of baseline, p<0.05 from no tone, 2-way ANOVA with Holm-Sidak post-hoc) and mPFCgamma (Fig 9c; 198.6 ± 48.7% of baseline, p<0.05 from no tone, 2-way ANOVA with Holm-Sidak post-hoc) without significantly altering evoked theta (Fig 8c; 132.0 ± 16.1 % of baseline,ns, 2-way ANOVA) or gamma (Fig 10c; 169.7 ± 20.0 % of baseline, ns, 2-way ANOVA) inthe vHipp. In contrast, the conditioned stimuli did not significantly alter oscillatory activity inMAM-treated rats and was thus significantly attenuated compared to saline-treated rats (Fig7d, mPFC theta 146.1 ± 18.2; Fig 9d, mPFC gamma 139.1 ± 12.0; Fig 10d, vHipp gamma116.4 ± 6.0, all p<0.05 from responses in control rats, 2-way ANOVA with Holm-Sidak post-hoc). There were no significant differences between saline and MAM rats in vHipp thetaactivity (Fig 8d, 124.4 ± 7.2; ns, 2-way ANOVA). It is important to note that there were nostatistically significant differences in gamma power preceding the onset of the tone in eitherthe mPFC or vHipp of conditioned MAM or saline rats.

Control rats that had been exposed to the tone prior to the conditioning period demonstrated asignificantly attenuated conditioned response to the tone (Fig 11). Specifically, a reducedmPFC oscillatory response was observed in the theta band (Fig 7e; 147.2 ± 19.8 % of baseline,p<0.05 from non-tone pre-exposed rats, 2-way ANOVA with Holm-Sidak post-hoc) whereasno significant effect was observed in mPFC gamma (Fig 9e; 146.5 ± 16.0 % of baseline, ns,2-way ANOVA) or vHipp theta (Fig 8e; 111.8 ± 8.8 % of baseline, ns, 2-way ANOVA) orgamma (Fig 10e; 147.6 ± 21.3 % of baseline, ns, 2-way ANOVA). Pre-exposure to theconditioned stimulus did not significantly alter oscillatory activity in MAM-treated rats (Fig7f, mPFC theta 130.9 ± 16.6; Fig 8f, vHipp theta 116.8 ± 12.3; Fig 9f, mPFC gamma 126.1 ±9.8; Fig 10f, vHipp gamma 127.2 ± 5.9, all ns, 2-way ANOVA).

DiscussionThese data demonstrate that MAM-treated rats display a pathological reduction in PVinterneuron density throughout the mPFC and vHipp that is associated with altered prefrontaland hippocampal activation during task performance. Furthermore, the degree of activation ofcortical assemblies was correlated with the behavioral response to a conditioned tone in a latent

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inhibition paradigm. Taken together, we propose that a decrease in intrinsic GABAergicsignaling may be responsible, at least in part, for the prefrontal and hippocampalhypofunctionality consistently disrupted in animal models as well as in schizophrenia patients.

A regionally selective decrease in PV interneuron density was observed throughout the medialprefrontal cortex and ventral (but not dorsal) subiculum of the hippocampus in MAM-treatedrats. This decrease appears specific for PV containing interneurons and is not associated withchanges in GAD-67 interneurons or total neuronal number (Moore et al., 2006). This isconsistent with previous reports demonstrating reduced PV interneuron functionalitythroughout the cortex, including dorsolateral prefrontal cortex, as well as throughout all of thesub-regions of the hippocampus in post-mortem brains of schizophrenia patients (Woo et al.,1998; Zhang and Reynolds, 2002; Lewis et al., 2005). A decrease in parvalbumin-containinginterneurons is also consistently observed across a variety of animal models of schizophrenia,including the MAM model (Penschuck et al., 2006), the chronic phencyclidine (PCP) model(Abdul-Monim et al., 2007), in rats reared in isolation (Harte et al., 2007) and the amygdalaactivation model (Berretta et al., 2004). It is important to note that the decrease in PVimmunoreactivity and mRNA expression noted in post mortem studies or animal models isbelieved to be associated with a reduced expression of PV rather than an actual decrease in thenumber of interneurons (Hashimoto et al., 2003).

Given their strong perisomatic innervation of glutamatergic neurons, parvalbumin interneuronsare situated to potently regulate pyramidal cell output (Lewis and Lund, 1990). Thus it is likelythat a reduction in PV interneuron functionality would interfere with the coordinated activityof cortical assemblies that drive synchronous activity in the gamma band across brain networks.Indeed, the present study demonstrates that MAM-treated rats display a deficit in the abilityto activate cortical and hippocampal assemblies during a latent inhibition paradigm. Thus,following tone-shock pairing in saline-treated rats, the conditioned tone induced a sustainedincrease in low frequency (theta) activity and a transient increase in high (gamma) frequencypower in the mPFC, without significantly altering coordinated activity in the vHipp. This isconsistent with previous work showing that fast-spiking interneurons display a robust, transientincrease in firing rate in response to a conditioned tone, whereas the response observed inpyramidal neurons is more sustained, being time-locked to the duration of CS presentation(Baeg et al., 2001). Furthermore, it has been suggested that the transient, high-frequencyactivation of interneurons may direct attention towards the CS while the more sustained,pyramidal cell activity may play a role in the anticipation of the unconditioned aversive stimuli(Baeg et al., 2001).

Interestingly, the present study demonstrated that the fear-conditioned tone did notsignificantly alter oscillatory activity in MAM-treated rats compared to the robust responsesobserved in saline-treated rats. Given the role for fast-spiking interneurons in the generationof gamma oscillatory potentials (Buzsaki et al., 1983; Whittington et al., 1995; Tamas et al.,2000; Bartos et al., 2002; Bartos et al., 2007; Freund and Katona, 2007), we suggest that thisis likely associated with the observed decrease in PV interneuron density. Indeed, the deficitin gamma activity in the MAM-treated rats was task-independent and observed in both themPFC and vHipp; regions that exhibited significant reductions in PV neuron density. Thesedata are consistent with studies demonstrating a clear correlation between PVimmunoreactivity and the generation of gamma rhythms in entorhinal cortical slices fromlysophosphatidic acid 1 receptor knockout mice as well as following ketamine administration(Cunningham et al., 2006). In addition, a disruption in the recruitment of PV-positive neuronshas been shown to alter pyramidal cell activity and oscillatory network function in a conditionalGluR-APVCre−/− knockout mouse (Fuchs et al., 2007). These alterations in network activitywere reportedly associated with impairments in spatial working memory and exploratorybehavior, suggesting that PV interneuron recruitment is required for the expression of key

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aspects of animal behavior (Fuchs et al., 2007). Indeed, the failure of a conditioned stimulusto evoke gamma activity in these regions in MAM-treated rats suggests that the ability toassociate a stimulus with an event, either neutral or noxious, is impaired.

Functional imaging studies show that schizophrenia patients consistently fail to activate themPFC and vHipp during cognitive tasks (Heckers et al., 1998; Meyer-Lindenberg et al.,2001; Perlstein et al., 2001; Weiss and Heckers, 2001; Barch et al., 2003; Preston et al.,2005), and abnormalities have been reported in induced theta- and gamma-band activity infrontal regions during a number of different paradigms, including cognitive processes such asworking memory (Haig et al., 2000; Ford et al., 2002; Gonzalez-Hernandez et al., 2003;Spencer et al., 2003; Schmiedt et al., 2005; Cho et al., 2006; Basar-Eroglu et al., 2007).Moreover, these deficits in synchronous activity are significantly correlated with reduced taskperformance in schizophrenia patients, further demonstrating the role for coordinated networkactivity in the expression of cognitive behaviors. Consistent with this premise, we now reportthat deficits in mPFC and vHipp oscillatory activity are associated with an impaired behavioralexpression of latent inhibition in MAM-treated rats. Thus, control rats that had been exposedto the tone prior to the conditioning period demonstrated a significantly attenuated prefrontaloscillatory response to the tone in both the theta and gamma frequencies that was correlatedwith a robust latent inhibition, i.e. an attenuated conditioned response to the tone. In contrast,MAM-treated rats displayed a deficit in the behavioral expression of latent inhibition, withprevious exposure to the tone having no significant effect on the locomotor response to theconditioned stimulus (Flagstad et al., 2005). We found that this was correlated with an inabilityto activate cortical assemblies in response to the conditioned tone in MAM-treated rats.

Taken together, the data presented here are consistent with evidence for a reduction inprefrontal and hippocampal activation during task performance in schizophrenia and animalmodels of psychosis. Of course, these studies focus primarily on changes in neuronal activityduring task performance that does not necessarily reflect the baseline activity of the structure.Indeed, there is significant and increasing evidence for enhanced activity within thehippocampus of schizophrenia patients at rest (Heckers et al., 1998; Malaspina et al., 1999;Medoff et al., 2001; Lewandowski et al., 2005; Malaspina et al., 2008; Tamminga et al.,2008).

Evidence for baseline hippocampal hyperactivity was first reported in the medial temporal lobein human schizophrenia patients using SPECT imaging (Malaspina et al., 1999). More recently,techniques with higher spatial resolution have provided evidence for increased regionalcerebral blood flow (H2015-PET: (Tamminga et al., 2008)) and increased regional cerebralblood volume (dynamic susceptibility contrast MRI: (Malaspina et al., 2008)) at rest inschizophrenia patients. Furthermore, the hyperactivity within these hippocampal subfields ishighly correlated with clinical measures of psychosis and delusions (Malaspina et al., 2008;Tamminga et al., 2008) suggesting that aberrant hippocampal activity may be a key anatomicalsubstrate involved in the positive symptoms of schizophrenia.

We reported previously that a pathologically enhanced activity within the vHipp actually drivesthe dopamine dysfunction in MAM-treated rats (Lodge and Grace, 2007). Thus, MAM-treatedrats demonstrate increased DA neuron population activity (i.e. the number of neurons firingspontaneously). This is attributed to hyperactivity within the ventral hippocampus since TTXinactivation of the vHipp normalized both the augmented DA neuron activity and behavioralhyper-responsivity to amphetamine administration (Lodge and Grace, 2007). Given thepathological decrease in PV neuron density within the vSub reported in the current study, wepropose that a decrease in intrinsic GABAergic function may be the origin of the hippocampalhyperactivity purported to underlie the dopamine dysfunction in psychosis (Lodge and Grace,2007; Lisman et al., 2008).

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The data presented here are consistent with evidence for a reduction in PV-containinginterneurons that is correlated with altered prefrontal and hippocampal activation during taskperformance in schizophrenia and animal modes of psychosis. Furthermore, the inability toactivate cortical assemblies is consistent with the impairments in cognitive function that are ahallmark of the disease. As we have emphasized previously (Lodge and Grace, 2007), we areaware that gestational MAM administration is not necessarily an accurate recapitulation of theetiology of schizophrenia in humans. Nonetheless, we posit that at the core of this disorder isa disruption of systems interactions that can be modeled in animals, but when placed in thecontext of complex human brain and behavioral patterns, yields the complex pattern ofpsychopathology recognized as schizophrenia. Such an understanding of the functionalinteractions among these systems and how disruption within these circuits affects informationprocessing is central to gaining a better understanding of disease. Moreover, these resultssuggest that a more effective pharmacotherapeutic strategy may lie in the restoration ofinterneuron regulation of patterned activity within ventral hippocampal circuits.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgementsThe authors would like to thank Niki MacMurdo & Emily Mahar for their valuable contributions, Witold Lipski forhis assistance with MATLAB, and Brian Lowry for the production, development and support of the custom designedelectrophysiology software (Neuroscope). This work was supported by the USPHS MH57440 (AAG) and a YoungInvestigator Award from NARSAD - The Mental Health Research Association (DJL)

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Figure 1.Summary of combined behavioral testing and stimulus-evoked local field potential (LFP)recordings in a latent inhibition paradigm. Rats were divided into 2 groups, with one grouppre-exposed to a non-salient tone and the other group having no tone presentation. All ratswere subsequently exposed to a standard auditory fear conditioning procedure that involved atone presentation that co-terminated for the final second with a mild footshock. 24 hoursfollowing conditioning, rats were placed in the conditioning chamber where the locomotor andtone-evoked responses to the conditioned tone were recorded.



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Figure 2.Histological localization of electrode sites within the prefrontal cortex and hippocampus.Numbers beside each plate represent approximate A/P distance from Bregma.



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Figure 3.MAM-treated rats display a regionally specific reduction in the density of parvalbumin-positiveneurons throughout the medial prefrontal cortex (mPFC) and the ventral subiculum of thehippocampus (vSUB). Confocal z-stack images of parvalbumin (red) and GAD67 (green)stained sections throughout the prelimbic sub-region of mPFC (A) and vSub (B) demonstratea decrease in PV interneurons. The density of parvalbumin and GAD-67 positive/parvalbuminnegative neurons are depicted in (C) and (D) respectively. * represents statistically significantdifference from control (prenatal saline administration) (p<0.05 1-way ANOVA, Tukey post-hoc n = 4–5 rats/group). Scale bars represent 50µm for all.



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Figure 4.Spontaneous local field potential oscillations throughout the ventral hippocampus of saline-(A,B) and MAM- (C,D) treated rats. A time-domain reconstruction (A,C) demonstrates thepredominant theta rhythm (blue line), a hallmark of oscillatory hippocampal activity, overlaidon a 2 second epoch of spontaneous activity (red line). The arrows indicate periods of highfrequency oscillations ‘riding’ the theta wave. A continuous wavelet time-frequency spectrum(B,D) demonstrates the power (Time-Integral Squared Amplitude: TISA), indicated by the redgradient contour, across frequency for a 4 minute period (y1 axis). The global wavelet spectrumfor the entire time range is depicted by the yellow line, plotted against the y2 axis.



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Figure 5.Spontaneous local field potential oscillations throughout the medial prefrontal cortex of saline-(A,B) and MAM- (C,D) treated rats. A time-domain reconstruction (A,C) demonstrates a lowfrequency (delta) rhythm (blue line) overlaid on a 2 second epoch of spontaneous activity (redline). Strong periodic high frequency oscillations (~20Hz) are present in the raw trace. Acontinuous wavelet time-frequency spectrum (B,D) demonstrates the power (Time-IntegralSquared Amplitude: TISA), indicated by the red gradient contour, across frequency for a 4minute period (y1 axis). The global wavelet spectrum for the entire time range is depicted bythe yellow line, plotted against the y2 axis.



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Figure 6.MAM-treated rats display deficits in latent inhibition. Rats that had received a mild footshockpaired with a tone display a robust decrease in locomotor activity in response to the conditionedtone (dark bars). Control rats (A) that had been exposed to the tone prior to the conditioningperiod displayed robust latent inhibition, i.e. an attenuated locomotor response to the tone (A,light bars). In contrast, MAM-treated rats (B) display a deficit in latent inhibition with theprevious exposure to the tone having no significant effect on the locomotor response to theconditioned stimulus (B, light bars). * represents significant difference compared to baselinewhereas † represents significant difference between tone and no tone pre-exposure (p<0.05 2-way RM ANOVA with Holm-Sidak post hoc: n = 5 rats/group).



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Figure 7.mPFC theta response to the conditioned tone is reduced in MAM-treated rats. Multi-taperspectral analyses of tone-evoked local field potential responses demonstrate that tonepresentation alone induces a mild increase in prefrontal theta (4–12Hz) oscillations in bothsaline (A) and MAM (B) rats. Control rats that had received a mild footshock paired with atone display a massive and sustained increase in theta activity evoked by the conditioned tone(C), that is attenuated in rats that had been exposed to the tone prior to the conditioning period(E). In contrast, MAM-treated rats did not display a robust response to the conditioned toneeither in rats with (F) or without (D) previous tone exposure. The horizontal line depicts the 2



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second tone presentation whereas the dashed line represents the control response (i.e. no tonepresentation); n = 3–5 rats/group.



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Figure 8.vHipp theta is unaltered following fear conditioning. Multi-taper spectral analyses of tone-evoked local field potential responses demonstrate that tone presentation alone has nosignificant effect on hippocampal theta (4–12Hz) oscillations in saline (A) and MAM (B) rats.Interestingly there were no significant changes in theta activity to the conditioned tone in eithercontrol rats (C & E) or MAM-treated rats (D & F). The horizontal line depicts the 2 secondtone presentation whereas the dashed line represents the control response (i.e. no tonepresentation); n = 3–5 rats/group.



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Figure 9.High frequency mPFC responses to the conditioned tone are reduced in MAM-treated rats.Multi-taper spectral analyses of tone-evoked local field potential responses demonstrate thattone presentation alone induces a mild increase in prefrontal gamma (30–55Hz) oscillations insaline (A), but not MAM (B), rats. Control rats that had received a mild footshock paired witha tone display a transient but significant increase in high frequency activity evoked by theconditioned tone (C), that is attenuated in rats that had been exposed to the tone prior to theconditioning period (E). In contrast, MAM-treated rats did not display a robust response to theconditioned tone either in rats with (F) or without (D) previous tone exposure. The horizontal



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line depicts the 2 second tone presentation whereas the dashed line represents the controlresponse (i.e. no tone presentation); n = 3–5 rats/group.



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Figure 10.The high frequency responses to the conditioned tone are blunted in the vHipp of MAM-treatedrats. Multi-taper spectral analyses of tone-evoked local field potential responses demonstratethat tone presentation alone induces a mild increase in hippocampal gamma (30–55Hz)oscillations in both saline (A) and MAM (B) rats. In contrast to that observed in the mPFC,neither saline nor MAM-treated rats displayed a robust response to the conditioned tone eitherin rats with (E,F) or without (C,D) previous tone exposure. However, a significant reductionin tone-evoked oscillatory activity was observed in MAM-treated rats. The horizontal linedepicts the 2 second tone presentation whereas the dashed line represents the control response(i.e. no tone presentation); n = 3–5 rats/group).



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Figure 11.MAM-treated rats display a significantly attenuated oscillatory activity during latent inhibitiontraining and testing. Analysis of the maximum increase in oscillatory activity demonstratessignificant deficits in the induction of mPFC theta (A) (4–12Hz), mPFC gamma (C) (30–55Hz),vHipp gamma (D) (30–55Hz), but not vHipp theta (B) (4–12Hz) between saline and MAM-treated rats. * represents significant difference compared to no tone trials, ‡ representssignificant difference between tone (dark grey bar) and no tone (light grey bar) pre-exposure,and † represents significant difference between saline and MAM-treated rats (p<0.05 2-wayANOVA with Holm-Sidak post-hoc: n = 3–5 rats/group).



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